"Silage is the material produced by the controlled fermentation of a crop of high moisture content": (Chemistry and Biochemistry of Herbage, edited by G. W. Butler and R. W. Bailey, Academic, Press, London and New York, Vol. 3, 1973, pp. 33-37, Chapter 28 entitled "The Ensilage Process", authored by P. McDonald and R. Whittenbury). Fermentation of silage incudes production of organic acids by bacteria, often Lactobacillus which are naturally present on fresh herbage or due to direct addition of Lactobacillus, or addition of acids or other preservatives. Herbage, for purposes of this application, is used to refer to all crop materials which are fermented into silage. Only preservatives which do not interfere with the desired Lactobacillus fermentation can be used in silage when the organic acids are produced by fermentation. Silage is an extremely high moisture product and is most efficiently stored in a hermetically sealed container.
Silage can be spoiled microbiologically under both aerobic and anaerobic conditions. If "oxygen is in contact with herbage which is being converted to silage, then aerobic microbial activity occurs and the material becomes a useless, inedible, and frequently toxic product" (Chemistry and Biochemistry of Herbage). Because oxygen often cannot be sufficiently excluded, it is desirable to utilize other means to prevent microbial spoilage. If anaerobic conditions can be produced, then spoilage may still readily occur due to "clostridial fermentation which leads to the production of carbon dioxide, ammonia and undesirable nitrogenous compounds such as amines". One way to decrease clostridial activity is to produce silage with 28% or more dry matter. However, silage as a final product often contains less than 28% dry matter and usually contains less than 28% dry matter when fermentation is initiated. In those cases where silage contains 28% or more dry matter, yeast and mold fermentation can still continue to occur, and Lactobacillus may grow in up to as much as 70% dry matter. This latter fermentation is desirable if it involves only Lactic acid producing bacteria since the lactic acid produced lowers the pH of the silage which acts to preserve the silage. The mold and yeast fermentations are undesirable as they produce spoilage. Similar spoilage problems due to mold and yeast fermentations exist for hay and mixed feeds which are often stored while containing more than 10% moisture.
The production of silage is, in itself, testimony to the need to preserve animal feeds. However, spoilage of silage is clearly contradictory to its reason for production. The wetter the silage the more difficult it is to preserve utilizing fermentation because the critical pH for preservation must be lower in those cases when silage is wetter. A standard method of preservation of hay today includes application of propionate compounds to the hay in amounts of 10 pounds per ton of hay which is 0.5%. However, propionates are not used in silage as they would interfere with the growth of Lactobacillus which are important for production of acids to lower pH. Thus, the compound which is known to preserve against spoilage organisms is also inhibitory to desirable organisms.
Maleic acid and fumaric acid and their esters have been known for a long time. In 1940, U.S. Pat. No. 2,218,181 to Searle and Tisdale describe the use of said esters for control of bacteria, fungi, and insects. They found the esters to have broad activity against such diverse organisms as Fomes annosus, Ceratostomella pilifera, Penicillium digitaum, mixed lumber molds, Aspergillus niger, Penicillium expansu, and Bacillus mesentericus. They found dimethyl fumarate (DMF) to be effective against bacteria and molds which decompose leather and proteolytic bacteria which attack dry casein powder. Further, in 1982, U.S. Pat. No. 4,346,118 issued to Mir N. Islam teaches that di-alkyl esters of fumaric acid have surprisingly strong antimicrobial activity with a broad spectrum against various microorganisms including bacteria, fungi, and yeast. Some bacteria inhibited by dimethyl fumarate in concentrations ranging from 0.001-0.01% are Lactobacillus acidophilus, Lactobacillus casei and Lactobacillus plantarum (lines 55-60, Col. 4, U.S. Pat. No. 4,346,118). These are the very type of microorganisms which produce silage (Chemistry and Biochemistry of Herbage).
Islam suggested that microbicidal amounts of di-alkyl esters of fumaric acid could be used to preserve a variety of "food", "feed" and "agricultural crops" including such agricultural crops as "cereal granis, legumes, oil seeds, nut seeds, dried fruits, tubers, root crops, silage, green wood (lumber), wood chips, wood pulp, canes; forage crops, flower bulbs; crop by-products such as citric pulp, apple pampas, almond hulls, etc." Addition of DMF to silage requires, by definition, that DMF be added after fermentation is complete. Without the fermentation being complete, a product cannot be called silage. DMF cannot be added prior to fermentation of herbage into silage (un-cured herbage), especially not in microbicidal concentrations, because DMF inhibits the desirable fermentation organisms. If the fermentation organisms are inhibited, then silage will not be produced. Islam (U.S. Pat. No. 4,346,118) shows that as little as 0.25 ounces of DMF per 100 pounds of flour which is combined with other ingredients (Table data, Example III, l 35-45) to produce only 0.0029% DMF in bread, inhibits natural bread fermentation. In fact, an "initial `sponge` dough was prepared omitting the preservative to facilitate (the desirable) yeast fermentation". In spite of this, there was still a fermentation inhibition. These findings are consistent with the teaching that 0.001-0.01% DMF inhibits silage fermentation organisms and must be added after the fermentation is complete to avoid inhibition of the desired fermentation.
Most examples in Islam U.S. Pat. No. 4,346,118 utilize 0.2% or more DMF, which is the concentration claimed in U.S. Pat. No. 4,346,118. The only example of lower concentrations used by Islam are as described above for bread or were used in a very short term study (48 hours) using plating media with a very dilute human infection mold inoculum. (Example XII-Column 12) These 48 hr data are not useful teaching for preservation of animal feeds for prolonged time periods (months and years) where ester hydrolysis degradation of DMF occurs and where a heavy mold "load" or infection occurs in a field and during harvesting.
Thus, it is clear that the di-alkyl esters of fumaric acid inhibit mold but could not be useful in un-cured herbage which is to be converted into silage by fermentation due to its reported bactericidal effects. Islam also points out that the concentrations at which di-alkyl esters of fumaric acid are applied to prevent fungus and mold growth depend upon the type of organic material being treated, its moisture content, the temperature and humidity and the period over which the preservation is desired. "The higher these parameters the greater would be the need for fungicide" (Column 5, lines 40-41). This may be why DMF can inhibit mold in bread in low concentrations, because baking not only lowers the amount of available water, but also kills mold such that the only source of mold for spoilage is the few spores that fall on bread during packaging or after being opened. In silage where humidity is extremely high, the spoilage organism "load" is very high from the field, and storage periods are quite long (months or even years). The writings of Islam teach that high concentrations of di-alkyl esters would be needed for preservation, perhaps up to 10% by weight of the material being treated (Col. 5, lines 35-43), and addition would have to occur after fermentation from herbage into silage is complete.
The broad spectrum anti-microbial activity of esters of fumaric acid is further suggested by two other publications. Gershom and Shanks ("Antifungal Properties of 2-Bromo-3-Fluorosuccinic Acid Esters and Related Compounds", Journal of Medicinal Chemistry, 1977, Vol. 20, No. 4, pp 606-609) shows that a variety of esters of fumaric acid are inhibitory to a variety of molds and yeast. Sahajawalla and Ayres reported that dimethyl fumarate inhibited all species of yeast, bacteria and molds against which it was tested in their effects to develop a new antimicrobial substance for use in people. Some of the organisms inhibited include a Streptococcus mutans and Streptococcus sanguis which are part of the Streptococcus family important in silage fermentation. (American Association of Pharmaceutical Scientists, First National Meeting and Premiere Exposition, The Washington Hilton, Washington, D.C., Nov. 2-6, 1986, abstracts of contributing papers, page 92S, No. 210). They further reported that dimethyl fumarate is not stable in aqueous environment. In water at 85.degree., DMF degraded completely within a week, and only 72% DMF remained intact at 45.degree. at the end of 8 weeks. In the presence of enzymes, DMF was even less stable with 50% degrading in less than 3 hours in human plasma and in less than 10 minutes in rabbit blood. In vaginal secretions 50% degraded in 21 hours and in normal saline solution (which does not contain enzymes but does contain dissolved salts) 50% was degraded in 53 hours. Thus, it is clear that these findings of DMF instability in aqueous environment are consistent and compatible with the report of Islam that the efficacy of di-alkyl esters of fumaric acid depend upon the moisture content, temperature, humidity, and period over which preservation is desired.
We have now unexpectedly discovered that dimethyl fumarate (DMF) can be applied to high moisture hay or grains or other herbage prior to fermentation into silage by Lactobacillus or Pediococcus or Streptococcus or other microorganisms added or naturally present, with the DMF in sufficiently high concentrations which have been reported to inhibit silage producing microbes, but never-the-less the silage fermentation unexpectedly does occur; and the DMF is in a concentration expected to be too low to inhibit spoilage in these high moisture feeds but spoilage of the silage by molds and yeasts is unexpectedly prevented for long periods of time. Greater than 15% moisture would certainly be expected to degrade dimethyl fumarate and decrease its effect as reported by Islam (U.S. Pat. No. 4,346,118). High moisture refers to 15% moisture or greater for this invention. Silage often contains 45-80% moisture.
Islam (Inhibition of Mold in Bread by Dimethyl fumarate, Journal of Food Science, Vol. 47, 1982, pp 1710-1712) points out that the safety of dimethyl fumarate for use as a food additive must be established before it can be used in foods for people. The degradation products of dimethyl fumarate are fumaric acid and methanol. While fumaric acid is safe in humans, methanol is a relatively toxic compound. However, in ruminants, methanol is a normal constituent of the ruminant contents and varies from about 23-28 mg/ml on average (occasionally exceeding 100 mg/ml) in cows (Ruminant Methanol In Vivo and In Vitro, Journal of Dairy Science, Vol. 53, No. 10, pp 1511-1514). The methanol is normally present due to enzymatic breakdown of pectin in both hay and grain. Since the ruminant content of a medium size cow is about 40 gallons (160 liters), that would be about 4,000 mg on average up to 16 grams (in some cows) of methanol. For a 400 kg cow, that would be a "dose" of 10 to 40 mg/kg of methanol normally present at the exact moment the concentration of methanol in the rumen was determined. This is however, only a very small fraction of the methanol actually produced in the ruminant since it is metabolized rapidly by methanol utilizing bacteria present in the ruminant. Two of these bacteria are Eubacterium limosum and Methanosarcina ("Features of Rumen and Sewage Sludge Strains of Eubacterium Limosum, a Methanol-and H.sub.2 CO.sub.2 -Utilizing Species", Genthner, Davis, and Bryant, Applied Environmental Microbiology, Jul. 1981, page 12-19). Thus, silage or feed containing dimethyl fumarate for a 400 kg heifer who would eat about 9 kg of feed per day as dry matter (Nutrient Requirements of Dairy Cattle, Nutrient Requirements of Domestic Animal, National Academy of Sciences, 5th revised addition, 1978, pp 30-34) would produce a very small amount of methanol compared to the methanol normally present. Thus, although there may be some concern for introduction of dimethyl fumarate into foods to be consumed directly by humans, introduction of dimethyl fumarate into the food chain as an additive on ensilage wherein the dimethyl fumarate would be converted to fumaric acid and methanol is no problem, and may be beneficial to the ruminant. In the ruminant, there is no concern for safety because the end products are fumaric acid which is nontoxic in either animals or people and methanol which is rapidly cleared to nontoxic carbon dioxide by the bacteria present.